How neurons in a complex nervous system become correctly wired is a key question of developmental neurobiology. Identifying molecules involved in axon pathfinding and understanding how these molecules function individually and in concert to guide axons through a complex cellular environment is currently the central focus of the field. The experiments outlined here address the guidance molecule Commissureless, which is absolutely required for axons to cross the CNS midline of Drosophila embryos. A multidisciplinary approach will be undertaken to screen for and identify crucial proteins that interact with Commissureless: Proposed are expression cloning and biochemical techniques to identify proteins that specifically interact with the extracellular domain of Commissureless; and a genetic screen for dosage sensitive modifiers of commissureless that can potentially identify several components of a commissureless dependent pathway. The specific aims are to: la) Identify proteins that interact with the extracellular domain of Commissureless as it guides axons across the CNS midline; lb) Isolate, map and clone the gene (or genes) that may function as a significant partner or "receptor" for Commissureless and characterize the partner protein and its mechanism of interaction with Commissureless biochemically and molecularly; 2) Identify genes that interact with commissureless by screening for dosage sensitive modifiers of commissureless using two variants of a second site enhancer of lethality screen. The proposed experiments will contribute significantly to our understanding of the fundamental mechanisms and molecules associated with growth cone guidance.